RESUMO
Genetic and molecular approaches have been critical for elucidating the mechanism of the mammalian circadian clock. Here, we demonstrate that the ClockΔ19 mutant behavioral phenotype is significantly modified by mouse strain genetic background. We map a suppressor of the ClockΔ19 mutation to a â¼900 kb interval on mouse chromosome 1 and identify the transcription factor, Usf1, as the responsible gene. A SNP in the promoter of Usf1 causes elevation of its transcript and protein in strains that suppress the Clock mutant phenotype. USF1 competes with the CLOCK:BMAL1 complex for binding to E-box sites in target genes. Saturation binding experiments demonstrate reduced affinity of the CLOCKΔ19:BMAL1 complex for E-box sites, thereby permitting increased USF1 occupancy on a genome-wide basis. We propose that USF1 is an important modulator of molecular and behavioral circadian rhythms in mammals. DOI:http://dx.doi.org/10.7554/eLife.00426.001.
Assuntos
Fatores de Transcrição ARNTL/metabolismo , Proteínas CLOCK/metabolismo , Relógios Circadianos , Ritmo Circadiano , DNA/metabolismo , Mutação , Fatores Estimuladores Upstream/metabolismo , Fatores de Transcrição ARNTL/genética , Animais , Sítios de Ligação , Ligação Competitiva , Proteínas CLOCK/genética , Relógios Circadianos/genética , Ritmo Circadiano/genética , Elementos E-Box , Regulação da Expressão Gênica , Genótipo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/metabolismo , Transdução de Sinais , Especificidade da Espécie , Fatores de Tempo , Transcrição Gênica , Ativação Transcricional , Fatores Estimuladores Upstream/genéticaRESUMO
Most laboratory mouse strains including C57BL/6J do not produce detectable levels of pineal melatonin owing to deficits in enzymatic activity of arylalkylamine N-acetyltransferase (AANAT) and N-acetylserotonin O-methyl transferase (ASMT), two enzymes necessary for melatonin biosynthesis. Here we report that alleles segregating at these two loci in C3H/HeJ mice, an inbred strain producing melatonin, suppress the circadian period-lengthening effect of the Clock mutation. Through a functional mapping approach, we localize mouse Asmt to chromosome X and show that it, and the Aanat locus on chromosome 11, are significantly associated with pineal melatonin levels. Treatment of suprachiasmatic nucleus (SCN) explant cultures from Period2(Luciferase) (Per2(Luc)) Clock/+ reporter mice with melatonin, or the melatonin agonist, ramelteon, phenocopies the genetic suppression of the Clock mutant phenotype observed in living animals. These results demonstrate that melatonin suppresses the Clock/+ mutant phenotype and interacts with Clock to affect the mammalian circadian system.